Phosphinothricin (PTC, 2-amino-4-methylphosphinobutyric acid) is an inhibitor of glutamine synthetase (GS). PTC is a “building block” of the antibiotic phosphinothricylalanylalanine. This tripeptide (PTT) is active against Gram-positive and Gram-negative bacteria and also against the fungus Botrytis cinerea. PTT is produced by the Streptomyces viridochromogenes strain Tü494, which is deposited in the Deutsche Sammlung für Mikroorganismen (German collection of microorganisms) under numbers DSM 40736 and DSM 4112 and which is obtainable from this source. It is known from German patent specification 2 717 440 that PTC acts as a total herbicide. The published application (EP-A-0257542) (corresponding U.S. Pat. No. 5,273,894) describes how a phosphinothricin N-acetyltransferase (pat) gene can be used to produce herbicide-resistant plants. The phosphinothricin N-acetyltransferase which is encoded by the pat gene modifies the PTC which appears intracellularly and detoxifies the herbicide.
The present invention now describes the use of deacetylase genes (dea), whose expression products are able to deacetylate N-acetylphosphinothricin (N-Ac-PTC) and/or N-Ac-PTT intracellularly, and thereby restore their antibiotic activity, for producing female-sterile plants.
An N-acetylphosphinothricin tripeptide deacetylase gene can be isolated from S. viridochromogenes Tü494. The dea gene is located downstream of the pat gene on the already known 4.0 kb BamHI fragment (EP-A-0 257 542) (corresponding U.S. Pat. No. 5,273,894). This gene is located on a BgIII/BamHI fragment and is fixed precisely by the sequence (FIG. 1 and Tab. 1). The protein sequence is defined by the DNA sequence.
An ATG codon, which is recognized in bacteria and plants, is used as the translation start codon; the Shine-Dalgarno sequence is underlined. This gene encodes the last step in the biosynthesis of PTT, i.e. the deacetylation of inactive N-acetylphosphinothricin tripeptide to give the active PTT.
It is known that the specificity of many enzymes is not restricted to one substrate. Thus, the phosphinothricin N-acetyltransferase which is encoded by the pat gene is actually used in PTT biosynthesis for acetylating desmethyl-PTC and, because of its lack of specificity, can be used for detoxifying PTC. By means of overexpressing the dea gene (using suitable promoters or by cloning onto high-copy vectors), an insufficiently specific N-acetyl-PTT deacetylase can now be employed for activating N-acetylphosphinothricin.
Other dea genes can be isolated from E. coli. Thus, it has been found that in E. coli, in contrast to other bacteria (e.g. rhizobias and streptomycetes), no activity can be detected in the so-called pat assay (dissertation of Inge Broer, University of Bielefeld Faculty of Biology, Expression des Phosphinthricin-N-Acetyltransferase-Gens aus Streptomyces viridochromogenes in Nicotiana tabacum (Expression of the Streptomyces viridochromogenes phosphinothricin N-acetyltransferase gene in Nicotiana tabacum), pp. 42-43, 1989) after the pat gene has been cloned into suitable expression vectors (Strauch et al., Gene, 63, 65-74, 1988; Wohlleben et al., Gene, 70, 25-37, 1988). In addition, when present in low copy number in E. coli, the pat gene is unable to confer resistance to PTT since the endogenous deacetylase nullifies the effect of the phosphinothricin N-acetyltransferase. Finally, this deacetylase activity can be demonstrated directly by the efficient inhibition of GS activity which occurs after adding N-acetylphosinothricin. The deacetylase converts N-Ac-PTC into PTC, which then inhibits the GS in a known manner, as can be measured in a γ-glutamyltransferase assay (Bender et al., J. Bacteriol. 129, 1001-1009, 1977). This is due to the possession by E. coli of an endogenous deacetylase activity.
This activity is apparently not present in the argE mutant which is known from the literature (Baumberg, Molec. Gen. Genetics 106, 162-173, 1970). Other E. coli deacetylase mutants are easy to select: following classical (Delić et al., Mut. Res. 9, 167-182, 1970; Drake and Baltz, Ann. Rev. Biochem. 45, 11-38, 1976) or Tn5 mutagenesis (Kleckner, Ann. Rev. Genet. 15, 341-404, 1981), such mutants can be recognized on PTT-supplemented minimal medium by the fact that it is only they which are able to grow after having been transformed with a pat gene which is cloned into a low copy number vector.
The E. coli deacetylase gene can therefore be isolated by using conventional methods (Maniatis et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982) to construct a gene library in, for example, the E. coli argE mutant or in a freshly isolated mutant.
Methods for isolating other deacetylase genes can be inferred from that which is described above: e.g. isolating new organisms which are PTT-sensitive despite the presence of a pat gene on a low copy number vector, and subsequently isolating a deacetylase gene.